离体鸡肝细胞核中卵黄蛋白原基因的体外RNA合成与表达
In vitro RNA synthesis and expression of vitellogenin gene in isolated chicken liver nuclei.
作者信息
Panyim S, Ohno T, Jost J P
出版信息
Nucleic Acids Res. 1978 Apr;5(4):1353-70. doi: 10.1093/nar/5.4.1353.
Abstract
Optimal conditions for prolonged in vitro synthesis of RNA in isolated chicken liver nuclei have been described. It is shown by incorporation of gamma32P-GTP into RNA, analysis of the product on sucrose density gradient, and digestion with alkaline phosphatase and ribonuclease A that there is reinitiation of RNA synthesis. Polynucleotide kinase activity has been ruled out as explanation for the incorporation of gamma32P-GTP. alpha-Amanitin inhibits RNA synthesis by about 50%. Nuclei prepared from estradiol-treated chicks have twice the RNA synthesis activity as the controls. RNA is synthesized in the presence of Hg-UTP and the mercurated product separated by affinity chromatography on sulfhydryl-Sepharose column under stringent conditions. Vitellogenin mRNA sequences are measured by hybridization with DNA complementary to vitellogenin mRNA. Estradiol treatment leads to a 10-fold increase in vitellogenin mRNA sequences.
摘要
已描述了在离体鸡肝细胞核中长时间体外合成RNA的最佳条件。通过将γ32P-GTP掺入RNA、在蔗糖密度梯度上分析产物以及用碱性磷酸酶和核糖核酸酶A消化表明存在RNA合成的重新起始。多核苷酸激酶活性已被排除作为γ32P-GTP掺入的解释。α-鹅膏蕈碱抑制RNA合成约50%。从经雌二醇处理的雏鸡制备的细胞核具有两倍于对照的RNA合成活性。在Hg-UTP存在下合成RNA，并在严格条件下通过巯基-琼脂糖柱上的亲和色谱分离汞化产物。通过与卵黄蛋白原mRNA的互补DNA杂交来测量卵黄蛋白原mRNA序列。雌二醇处理导致卵黄蛋白原mRNA序列增加10倍。
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